Different Levels Of HER2 Expression Research Articles

Abstract 3135: BSI-730, a bispecific antibody targeting HER2 and PD-L1 for the development of a first-in-class bispecific ADC

Abstract Background: Antibody-drug conjugates (ADCs) have become a promising therapeutic option for patients with cancer. Bispecific ADCs targeting two tumor-associated antigens have potential differential anti-tumor advantages. HER2 and PD-L1 are co-expressed in some tumor cell types, such as breast cancer, gastric cancer, lung adenocarcinoma and urothelial carcinoma. A bispecific ADC targeting HER2 and PD-L1 should provide therapeutic benefit, particularly for patients with HER2 low cancer. Dual-target mediated cytotoxicity combined with PD-L1 mediated immunotherapy provides an innovative approach for cancer patients. Experimental procedures: An anti-PD-L1 monoclonal antibody was identified from A/J mice immunized with PD-L1-ECD-Fc and screened by the Biosion proprietary SynTracer® High Throughput Endocytosis Platform. A “knob-in-hole” bispecific antibody, BSI-730, targeting HER2 and PD-L1 was created with trastuzumab and the humanized anti-PD-L1 scFv. Target binding specificity, binding activity, and affinity of BSI-730 were evaluated by protein-based ELISA, cell-based FACS and Biacore-based SPR, and the ligand blocking activity of PD-L1 portion was measured by competitive ELISA and cell-based FACS. The internalization activities on various cancer cell lines with different expression levels of HER2 and PD-L1 were evaluated by using the SynTracer® Platform. Summary: BSI-730, a HER2/PD-L1 bispecific antibody, showed comparable binding affinity to HER2 as compared to trastuzumab, as well as comparable bioactivity to the parental anti-PD-L1 antibody regarding PD-L1 binding and PD-1/PD-L1 blocking. Based on a cell-based reporter assay, BSI-730 was able to reverse PD-L1 mediated T-cell suppression and exhibited comparable potency to the parental anti-PD-L1 antibody. The internalization of BSI-730 on HER2 low cancer cell lines was stronger than that of trastuzumab whereas the internalization of BSI-730 on HER2 high cancer cell lines was comparable to that of trastuzumab. Conclusion: BSI-730 is a novel bispecific antibody for the development of a first-in-class bispecific ADC with the potential advantage of increased potency that can be further investigated to treat HER2 and PD-L1 co-expression tumors, particularly HER2 low tumors. Citation Format: Xiaoyao Hao, Hongyan Li, Xiaodong F. Liu, Jinyu Liu, Wenwen Dai, Ying Pan, Hui-Han Hu, Jun Li, Shukai Xia, Qun Lyu, Hugh M. Davis, Mingjiu Chen, Zeyu Peng. BSI-730, a bispecific antibody targeting HER2 and PD-L1 for the development of a first-in-class bispecific ADC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3135.

Abstract 6337: Tri-specific CD3 x HER2 x HER2 antibodies in 1:2 format recognizing two different epitopes on HER2 demonstrate selective activity on human cancer cells with high HER2 expression

Abstract Human epidermal growth factor receptor 2 (HER2) is a membrane tyrosine kinase that was found to be amplified and overexpressed in various human cancers including breast cancer. Monoclonal antibodies targeting HER2 such as trastuzumab and pertuzumab provide good therapeutic benefits in patients with high HER2 expression tumors, however, not all patients respond to the treatment, and many suffer recurrence. There is still a large unmet need for targeted therapies to treat HER2 high expression tumors. T cell engaging antibodies that direct T cells to kill cancer cells by simultaneously binding to CD3 on T cells and tumor-associated antigens (TAA) on cancer cells have emerged as a promising new approach. Such therapeutics are potent, but safe use of them depends on selectivity of the TAA engaged. Though HER2 is highly overexpressed in ~ 20% of breast cancer, expression of HER2 is not restricted to cancer cells and present at low level in normal tissues. The capability of selectively targeting cancer cells over HER2 low expressing normal tissues is critical to the safety of CD3 x HER2 antibodies. It is noteworthy to mention that unlike antibody-drug conjugate (ADC) therapeutics, CD3 x HER2 antibodies might not be a good approach to target HER2 low/neg expression tumors due to the difference in mechanism of action. CMG6A19 is a 150 kD IgG-like tri-specific CD3 x HER2 x HER2 T cell-engaging antibody generated on Chimagen’s TRIAD platform. It is capable of simultaneously recognizing and binding to two different epitopes on HER2 proteins. Multiple human cell lines with different levels of HER2 expression were used as targets in the antibody-mediated hPBMC killing assays in vitro and in vivo, among which SK-BR-3, KPL-4 and BT474 have high HER2 expression as seen in HER2 positive cancers, HT55 and JIMT1 have medium level of HER2 expression, and PC3 and MCF7 have HER2 expression level that is close to in normal tissues. Conventional 1:1 IgG-like CD3 x HER2 bispecific antibodies were used as control. Results showed that CMG6A19 demonstrated higher tumor inhibition effect compared to control antibodies in SK-BR-3/KPL-4/BT474 models, meanwhile remarkably weaker effect in PC3/MCF7 models suggesting better safety in HER2 low expressing tissues. This selectivity of reactivity towards HER2 high expressing cells is likely due to the unique orientation of the antibody which allows better T cell activation when antigen abundance is high. Preliminary preclinical study in cynomolgus monkeys suggested that the antibody was well tolerated in monkeys at the dosage of 20 mpk and had a half-life that is comparable to conventional antibodies. CD3 x HER2 x HER2 tri-specific antibodies selectively showed potent anti-tumor activity on HER2 high expressing cells and good safety on HER2 low expressing cells. Current findings warrant further development in human. Citation Format: Jie Zhang, Xiaoqing Wang, Zhenhao Zhou. Tri-specific CD3 x HER2 x HER2 antibodies in 1:2 format recognizing two different epitopes on HER2 demonstrate selective activity on human cancer cells with high HER2 expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6337.

Methods for preparing tissue microarray slides using xenografts with different levels of HER2 expression to standardize HER2 detection.

Gene amplification and protein overexpression of human epidermal growth factor receptor type 2 (HER2) are specific targets for HER2-targeting drugs in breast, gastric, salivary gland, and colorectal cancers. The histopathological determination of HER2 status is crucial for treatment, highlighting the importance of improving HER2 detection accuracy in clinical practice. We prepared tissue microarray (TMA) slides for use as control slides for the standardization of gastric HER2 testing. Four human gastric cancer cell lines with HER2 scores of 3+, 2+, 1+, and 0 were xenografted in NOG mice. The TMA slides were constructed using samples from three different areas in these tumors. Staining properties were determined using six clinical kits for HER2. In TMA, HER2-positive tumors with HER2 scores of 3+ and 2+ showed good staining with all diagnostic kits, and the tissue images were similar to those of clinical samples. Xenograft tumor slides could potentially be used as external controls to standardize staining conditions for a variety of kits and may improve the accuracy of HER2 detection in clinical practice.

Correlation between different levels of HER2 expression in circulating tumor cells (cHER2 ratio) and metastatic behavior in stageIVaggressive breast cancer.

3036 Background: The presence of HER2 expressing (HER2+) circulating tumor cells (CTCs) occurs often in metastatic breast cancer (MBC) patients (pts). We have previously showed that the ratio among CTCs expressing high level of HER2 and the total number of HER2+ CTCs (circulating HER2 ratio, cHer2 ratio) has a prognostic role in MBC patients. Here we further investigate the role of the cHER2 ratio in the process of metastatic spread. Methods: Under IRB-approved study we prospectively analyzed blood samples of patients with MBC enrolled before starting a new line of therapy. Samples were collected from pts treated at Northwestern University (Chicago, IL) between 2016 and 2020. CTCs were enumerated through CellSearch (Menarini Silicon Biosystems, Bologna, Italy) and characterized for HER2 expression using the CellSearch CXC Kit. HER2+ CTCs were divided in 3 different categories (1+,2+,3+) leaning on fluorescence intensity. Pts with <5 CTCs (stage IV indolent) were excluded from the analysis. The cHER2 ratio, defined as the sum of 2+ CTCs and 3+ CTCs divided by the total number of HER2+ CTCs, was used to split the remaining pts in 2 different cohorts: cHER2 ratio high (> 0.75) (cHER2high) and cHER2 ratio low (≤0.75) (cHER2low). The frequency of each metastatic site (i.e. liver, lung, central nervous system, bone, lymph nodes, skin/soft tissue, serosa) and the total number of different sites involved (1-7, ≤2 and >2 sites) were compared among the two sub-populations and analyzed through Fisher exact test. Results: Out of 98 pts enrolled, 77 were classified as cHER2low and 21 as cHER2high. We observed a higher frequency of oligometastatic pts (≤2 sites involved) in the cHER2high cohort (16, 76%), compared to only 29 (37%) in the cHER2low (p<0.005). Moreover, the cHER2 ratio was associated with a tropism toward specific sites of disease spread with higher incidence of liver, lung and lymph nodes metastases in the cHER2low cohort (p<0.05). No other statistical associations were observed in respect of specific organ tropism. The frequency of involvement for each metastatic site among the two cohorts are reported in the table. Conclusions: Measuring CTCs enumeration and HER2 expression we identified two cohorts, cHER2high and cHER2low, associated with distinct patterns of metastatic spread. The cHER2low pts were correlated to multiple sites of metastatic involvement, with particular tropism toward liver, lung and lymph nodes. These results confirm the prognostic role of the cHER2 ratio, suggesting a peculiar biological meaning of the HER2+ 1+ CTCs.[Table: see text]

A DNA Nanoassembly-Based Approach to Map Membrane Protein Nanoenvironments

Super-resolution imaging has revealed that most proteins at the plasma membrane are not uniformly distributed but localize to dynamic domains of nanoscale dimensions. To investigate their functional relevance, there is a need for methods that enable comprehensive analysis of the compositions and spatial organizations of membrane protein nanodomains in cell populations. However, super-resolution methods are limited to analysing small, preselected subsets of proteins, at very low sampling fractions, and multiplexing analysis remains challenging. Here we describe thedevelopment of a non-microscopy based method for ensemble analysis of membrane protein nanodomains. The method, termed NANOscale DEciphEring of membrane Protein nanodomains (NanoDeep), is based on the use of DNA nanoassemblies to translate membrane protein organization information into a DNA sequencing readout. This method allows for detection of the inventory of proteins that forms the nanoenvironment of any reference membrane protein in cell populations. Using NanoDeep, we characterised the protein nanoenvironments surrounding Her2, a membrane receptor of critical relevance in cancer. We found that the occupancies of Her2, Her3 and EGFR in the nanoenvironments surrounding Her2 were similar in two cell lines with vastly different expression levels of Her2. Further, we found that adding Heregulin-β1 to cancer cells led to increased occupancy of Her2 and Her3, and to a lesser extent EGFR, in Her2 nanoenvironments. Importantly, we were able to modulate by design the inventory of proteins analysed by NanoDeep, including in the analysis of Her2 nanoenvironment two membrane proteins that have been reported to interact with Her2 (integrin α5β1 and CD63) and a protein unrelated to Her2 signalling (CD71). NanoDeep has the potential to provide new insights into the roles of the composition and spatial organization of protein nanoenvironments in the regulation of membrane protein function.

Investigating the Cytotoxic and Anti-proliferative Effects of Trastuzumab on MDA-MB-453 and MDA-MB-468 Breast Cell Lines With Different Levels of HER2 Expression

Introduction: Trastuzumab is a common treatment for HER2-positive breast cancer. Trastuzumab exerts its effect through inhibiting the intracellular signaling pathway induced by HER2. This study aimed to specifically investigate the cytotoxic effect of trastuzumab against two different breast cell lines, MDA-MB-453 (HER2-high) and MDA-MB-468 (HER2-low). Materials and Methods: The breast cancer cell lines were subjected to various concentrations of trastuzumab (1-1000 ng/mL). The trastuzumab’s effects were regularly monitored via direct observation by inverted microscopy. Effects of trastuzumab were determined on cytotoxicity, cell proliferation and apoptosis at 24 and 72 hours post-treatment via MTT colorimetric, cell cycle and apoptosis assays. Results: Microscopic observation demonstrated a dose-dependent increase in cell death at treated ones. The MTT assay showed that trastuzumab (1-1000 ng/mL) inhibited the growth of both cell lines in a dose-dependent manner. The findings of the present study revealed that trastuzumab induces a statistically higher cytotoxicity at all concentrations in MDA-MB-453 compared to MDA-MB-468 cells. It has been actually revealed that trastuzumab suppresses cell proliferation through inducing G1 phase arrest and triggers apoptosis in both cell lines. However, the effect of trastuzumab was found to be higher in MDA-MB-453, compared to MDA-MB-468. Conclusions: Trastuzumab could inhibit cell proliferation and trigger apoptosis in HER2-positive cells. Although Trastuzumab affected both cell lines, it significantly inhibited the cell growth of HER2-high cells.

99mTc-radiolabeled HER2 targeted exosome for tumor imaging

Exosomes represent unique features including nontoxicity, non-immunogenicity, biodegradability, and targeting ability that make them suitable candidates for clinical applications. Therefore, in this study, 99mTc-radiolabel HER2 targeted exosomes (99mTc-exosomes) were provided for tumor imaging. These exomes are obtained from genetically engineered cells and possessed DARPin G3 as a ligand for HER2 receptors. These exosomes were radiolabeled using fac-[99mTc(CO)3(H2O)3]+ synthon. The quality control showed high radiochemical purity (RCP) for 99mTc-exosomes (>96%). 99mTc-exosomes displayed a higher affinity toward SKOV-3 cells (higher HER2 expression) in comparison with MCF-7, HT29, U87-MG, A549 cell lines at different levels of HER2 expression. Trastuzumab (an antibody with a high affinity toward HER2) inhibited the binding of 99mTc-exosomes to SKOV-3 cells up to 40%. Biodistribution study in SKOV-3 tumor bearing nude mice confirmed the ability of 99mTc-exosomes for accumulation in the tumor. 99mTc-exosomes can visualize tumor in SKOV-3 tumor-bearing nude mouse. The blockage of HER2 receptors using trastuzumab (excessive amount) suggests the 99mTc-exosomes binding to the receptors and reduced the accumulation of 99mTc-exosomes in the tumor site. This suggest that 99mTc-exosomes interact with HER2 receptors and act through specific targeting.

Paclitaxel-Trastuzumab Mixed Nanovehicle to Target HER2-Overexpressing Tumors.

Paclitaxel is one of the most widely used chemotherapeutic agents thanks to its effectiveness and broad spectrum of antitumor activity. However, it has a very poor aqueous solubility and a limited specificity. To solve these handicaps, a novel paclitaxel-trastuzumab targeted transport nanosystem has been developed and characterized in this work to specifically treat cancer cells that overexpress the human epidermal growth factor receptor-2 (HER2). Methods: Alginate and piperazine nanoparticles were synthetized and conjugated with paclitaxel:β-cyclodextrins complexes and trastuzumab. Conjugated nanoparticles (300 nm) were characterized and their internalization in HER2-overexpressing tumor cells was analyzed by immunofluorescence. Its specific antitumor activity was studied in vitro using human cell lines with different levels of HER2-expression. Results: In comparison with free paclitaxel:β-cyclodextrins complexes, the developed conjugated nanovehicle presented specificity for the treatment of HER2-overpressing cells, in which it was internalized by endocytosis. Conclusions: It seems that potentially avoiding the conventional adverse effects of paclitaxel treatment could be possible with the use of the proposed mixed nanovehicle, which improves its bioavailability and targets HER2-positive cancer cells.

Molecular targeting of HER2-overexpressing biliary tract cancer cells with trastuzumab emtansine, an antibody-cytotoxic drug conjugate.

Trastuzumab emtansine (T-DM1) provides clinical benefit in breast cancers overexpressing human epidermal growth factor receptor 2 (HER2). However, its efficacy against biliary tract cancers (BTC) has not been evaluated. In this study, the effectiveness of T-DM1 in various BTC cell lines and xenograft models with different levels of HER2 expression was investigated. HER2 expression status in xenografts and patient tissue microarrays was assessed by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). Cell-surface HER2 expression levels and cell growth inhibition in response to T-DM1 were examined in 17 BTC cell lines. The antitumor activity of T-DM1 was evaluated in four xenograft mouse models with different levels of HER2 expression. The effects of T-DM1 on HER2 signaling, antibody-dependent cell-mediated cytotoxicity (ADCC), cell cycle, and apoptosis were assessed in vitro. Cell-surface expression of HER2 was observed in both gallbladder carcinoma and cholangiocarcinoma tissues. The anti-proliferative activity of T-DM1 was higher in BTC cell lines and breast cancer cell lines with higher levels of HER2 expression. The HER2 status (IHC score|HER2-to-CEP17 ratio by FISH testing) of each BTC xenograft was 3 +|8.3 for KMCH-1, 2 +|4.7 for Mz-ChA-1, 1 +/0|1.4 for OCUG-1, and 0|1.1 for KKU-100, and T-DM1 showed antitumor activity in proportion to the HER2 status. T-DM1 inhibited HER2 signaling and induced ADCC, mitotic arrest, and apoptosis in KMCH-1 cells. T-DM1 exhibited preclinical activity in HER2-overexpressing BTC. Further evaluation in clinical studies is warranted.

Concomitant Association of T-DM1 and Radiation: An In Vitro Study on HER2 Positive Cells of Breast Cancer

The better understanding of the EGFR receptors involved in the oncogenesis of HER2 positive breast cancers has led to the emergence of new therapeutic classes: the Antibodies Drug-Conjugate (ADC). Trastuzumab-DM1 (T-DM1) is an ADC combining the anti-HER2 monoclonal antibody trastuzumab with molecules of DM1, a powerful mitotic spindle inhibitor. In HER2 positive breast cancer, T-DM1 is a validated treatment but the effects of concomitant association with radiation are not known. The objective of this study is to determine in vitro the effects of concomitant irradiation with T-DM1 on five different HER2 positive breast cancer cell lines. Five cell lines of human breast cancer with different levels of HER2 expression (HCC-1954, BT-474, SKBR-3, MDA-MB-453, ZR-75). T-DM1 was provided by Roche®/Genetech upon MTA. Expression levels were determined by western blot. T-DM1 toxicity was assessed using the CellTiter-Glo® assay. Cell cycle analysis was performed by flow cytometry after incorporation of BrdU. HER2 cells were irradiated (GSR 137Cs 662 keV) at different dose levels (from 1 to 8 Gy) after exposure to T-DM1. Survival fractions were determined based on clonogenic assays or on cell survival after 5 doubling times. Radiosensitivity parameter (D10 and D37) were calculated using the mean values of a and b determined from the curves drawn for best fit to the experimental data. The action of T-DM1 on HER2 cells showed significant lethality by deprivation of the HER2 signaling pathway and intracellular DM1 action on the cell cycle with significant G2/M phase blocking. After 72h treatment with T-DM1 alone, the median effective dose (ED50) determined for the six cell lines used is function of the expression of the receptor HER2 except for one cell line (BT474). Using sub-toxic doses of T-DM1 and short-term drug exposure (6 or 12h), the cell viability still decreases dramatically after several days. After irradiation alone, D37 and D10 were significantly higher for the three high-HER2 expressing cells lines (HCC1954, BT474 and SKBr3) compared to the low-HER2 expressing cells (MDA-453 and ZR-75-1), with a linear increasing relationship between the radioresistance and the level of HER2 expression (D37: r2=0.9; p<0.0001 and D10: r2=0.7; p<0.0001). In combination with radiation, T-DM1 elicited strictly additive interaction. There was no significant difference on D10 compared to control for all cell lines. HCC1954 : D10 T-DM1 = 6.15±0.64 vs 6.0±0.24 for control; BT-474: D10 T-DM1 = 7.86±0.22 vs 7.6.0±0.43 for control; SKBr3 : D10 T-DM1 = 5.68±0.29 vs 5.43±0.14 for control; MDA-MB-453 : D10 T-DM1 = 4.82±0.07 vs 4.02±0.17 for control and ZR-75-1 = D10 T-DM1 = 3.3±0.3 vs 3.02±0.3 for control. Although HER2 expression is a radioresistant factor, T-DM1 did not have any radiosensitizing impact on HER2 positive breast cancer cells in this in vitro study. The major toxicity of T-DM1 alone could explain this phenomenon but further explorations are needed.

Preclinical study of antitumor activity of trastuzumab emtansine in HER2-positive biliary tract cancer.

256 Background: Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate composed of trastuzumab and DM1 (an anti-microtubule agent derived from maytansine) that is approved for the treatment of HER2-positive metastatic breast cancer. HER2-overexpression rate in biliary tract cancer (BTC) is reported as approximately 10%, and HER2-targeted therapy may enhance therapeutic efficacy in HER2-positive BTC. In this study, we investigated the antitumor activity of T-DM1 in several BTC cell lines and xenograft models. Methods: HER2 expression was detected by flow cytometry and Western blotting in 17 BTC cell lines. The cell growth inhibition activity of T-DM1 and DM1 was examined by quantifying the DNA with Hoechst 33258 nucleic acid stain. In vivo antitumor activity of T-DM1 was evaluated in three xenograft mouse models using cell lines with different levels of HER2 expression. HER2 expression in xenografts was assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), according to gastric cancer criteria. Cell cycle was assessed by flow cytometry after staining with propidium iodide. Apoptosis was assessed by caspase 3/7 activity measurement. Results: In the 17 BTC cell lines, HER2 expression was high in KMCH-1, Mz-ChA-1, and TGBC-18-TKB. Although cell growth inhibition of DM1 was similar in all cell lines (50% inhibitory concentration (IC50): 0.79–7.2 nM), that of T-DM1 was HER2-expression-dependent, with IC50 values in KMCH-1, Mz-ChA-1, and KKU-100 (which has low HER2 expression) of 0.031, 1.3, and 4.3 μg/mL, respectively. In KMCH-1 and Mz-ChA-1 xenograft models, T-DM1 treatment once every 3 weeks for 6 weeks showed significant dose-dependent antitumor activity, and tumor growth inhibition 21 days after first dose at 20 mg/kg T-DM1 was 108% (KMCH-1) and 75% (Mz-ChA-1), whereas there was no significant efficacy in KKU-100 xenograft model. Values for HER2 status (IHC score/FISH ratio of HER2 to CEP17) in KMCH-1, Mz-ChA-1, and KKU-100 tumor tissues were 3+/3.5, 2+/4.7, and 0/1.1, respectively. In KMCH-1, T-DM1 induced M phase arrest and apoptosis, as is reported in HER2-positive breast cancer cells. Conclusions: T-DM1 could be an effective therapy for HER2-overexpressed BTC.

Relationship between survival to breast cancer and level of HER2 expression by immunohistochemistry.

e13078 Background: Central review of NSABP B-31 showed that some central-review HER2-negative patients benefited from trastuzumab, but survival according to different levels of HER2 expression by immunohistochemistry (IHC) is unknown. The aim of the present study was to examine the survival of trastuzumab-naïve patients with breast cancer according to the level of HER2 expression by IHC. Methods: This was a retrospective study of all women who were treated for an invasive breast cancer at the Centre des maladies du Sein Deschênes-Fabia (Quebec City, Province of Quebec, Canada) between July 1999 and December 2010. Patients were grouped according to the HER2 status by IHC of their primary cancer (0 vs. 1+ vs. 2+ vs. 3+). Survival was obtained from the death registry of the Ministry of Health. Follow-up was censored on December 31st, 2012. Results: During the study period, 2571 patients with invasive breast cancer, with available HER2 status by IHC and survival data, and naïve to anti-HER2 therapy were treated at our center. Both 5- and 9-year overall survival (OS) decreased with increasing HER2 expression by IHC (9-year OS: 0: 86.2% vs. 1+: 71.3% vs. 2+: 73.9% vs. 3+: 69.3%; unadjusted log-rank test: P &lt; 0.0001; adjusted P = 0.04 for 3+). Both 5- and 9-year breast cancer-specific survival (BCSS) decreased with increasing HER2 expression by IHC (9-year BCSS: 0: 90.7% vs. 1+: 81.8% vs. 2+: 77.0% vs. 3+: 75.4%; unadjusted log-rank test: P &lt; 0.0001; adjusted P = 0.04 for 3+). Conclusions: Survival among trastuzumab-naïve breast cancer patients seems to follow a continuum across HER2 expression by IHC.

Bifunctional Toxin DARP-LoPE Based on the HER2-Specific Innovative Module of a Non-Immunoglobulin Scaffold as a Promising Agent for Theranostics

We have generated and characterized HER2-specific targeted toxin based on the low-immunogenic variant of Pseudomonas exotoxin A (LoPE), in which most of the human immunodominant B-cell epitopes have been inactivated. Nonimmunoglobulin DARPin-based HER2-specific protein was used as a targeting module for toxin delivery to the cellular target. Using confocal microscopy, it has been found that both domains in this hybrid toxin retained their functionality, i.e., the specific interaction with HER2 receptor, as well as the internalization and effective transport to ER typical of the wild-type Pseudomonas exotoxin A. The HER2-dependent cytotoxic effect correlated with receptor expression level at the cell surface, as shown in vitro using cell lines with different levels of HER2 expression. Due to the very high selective cytotoxicity against HER2-positive human tumor cells, as well as expected low immunogenicity, we believe that this new targeted toxin may be promising for future in vivo studies as a therapeutic agent for HER2-positive tumors.

New small 99mTc-labeled peptides for HER2 receptor imaging

The high expression of the human epidermal growth factor receptor 2 (HER2) and the accessibility of its extracellular domain make it an ideal target for the targeted delivery of anti-tumor drugs as well as imaging agents. In this study, the heptapeptide leucine-threonine-valine-serine-proline-tryptophan-tyrosine (LTVSPWY) as a new small peptide for an anti-HER2 target was labeled by incorporating 99mTc to the cysteine-based ligands CGGG (Cys-Gly-Gly-Gly) and CSSS (Cys-Ser-Ser-Ser) linked to this peptide. Both 99mTc-labeled peptides were evaluated for HER2 bindings as well as pharmacokinetics and tumor targeting. CGGG- and CSSS-LTVSPWY peptides were labeled with 99mTc using a gluconate ligand exchange. Cellular specific binding, affinities, and internalization of both peptides to the HER2 receptor were evaluated in the SKOV-3 cell line. Specific targeting of both peptides to the HER2 receptor was assessed in three cell lines with different levels of HER2 expression. Studies were performed in SKOV-3 tumor bearing mice for tumor targeting. Both peptides were labeled with 99mTc with more than 99% efficiency and showed favorable stability in solution and serum. The HER2 binding affinities of both the radiolabeled peptides were inhibited up to 60% by the unlabeled peptide, as well as with trastuzumab antibody. We observed nanomolar binding affinities for both radiolabeled peptides. The tumor uptakes were 4.95 ± 4.84% and 3.84 ± 2.53% for the CSSS and CGGG chelators, respectively, at 1 h after injection. However, tumor uptakes were similar for both peptides at 4 h postinjection, although a higher tumor to background ratio and lower radioactivity retention in the kidney were observed for CSSS, leading to a clearer tumor image with injection of this peptide. These small new peptides were selectively targeted to the HER2 receptor, and introduction of a serine residue into the chelator improved the pharmacokinetics of 99mTc labeled LTVSPWY for clear tumor imaging in animals.

Bioconjugation and Applications of Amino Functional Fluorescence Polymers.

Synthesis and novel applications of biofunctional polymers for diagnosis and therapy are promising area involving various research domains. Herein, three fluorescent polymers, poly(p-phenylene-co-thiophene), poly(p-phenylene), and polythiophene with amino groups (PPT-NH2 , PPP-NH2 , and PT-NH2 , respectively) are synthesized and investigated for cancer cell targeted imaging, drug delivery, and radiotherapy. Polymers are conjugated to anti-HER2 antibody for targeted imaging studies in nontoxic concentrations. Three cell lines (A549, Vero, and HeLa) with different expression levels of HER2 are used. In a model of HER2 expressing cell line (A549), radiotherapy experiments are carried out and results show that all three polymers increase the efficacy of radiotherapy. This effect is even more increased when conjugated to anti-HER2. In the second part of this work, one of the selected polymers (PT-NH2 ) is conjugated with a drug model; methotrexate via pH responsive hydrazone linkage and a drug carrier property of PT-NH2 is demonstrated on neuroblastoma (SH-SY5Y) cell model. Our results indicate that, PPT-NH2 , PPP-NH2 , and PT-NH2 have a great potential as biomaterials for various bioapplications in cancer research.

Abstract A147: MI130004, a new ADC with a payload of marine origin shows outstanding activity against HER2-expressing tumors

Abstract PM050489 is a tubulin-binding agent originally isolated from the marine sponge Lithoplocamia lithistoides. This compound binds with very high affinity to β-tubulin at a new site, disrupting the microtubule network and impairing its function during divison, which leads to mitotic aberrations. MI130004 (a novel antibody-drug conjugate formed by PM050489, a non-hydrolysable linker and trastuzumab) was tested for its in vitro and in vivo activity against selected tumor cell lines with different levels of HER2 expression. In vitro MI130004 showed very high potency and good selectivity for tumor cells that overexpressed HER2, namely HCC-1954 (IC50, 0.036 μg/mL), SK-BR-3 (IC50, 0.017 μg/mL) and BT-474 (IC50, 0.156 μg/mL). At the cellular level, MI130004 impaired tubulin polymerization, causing disorganization and disintegration of the microtubule network which ultimately led to mitotic failure. Expressing HER2 cells of breast (BT-474 and JIMT-1), gastric (Gastric-008 and N87) and ovarian (SK-OV-3 and A2780cis) as well as negative HER2 breast (MDA-MB-231) and gastric (Hs748t) were subcutaneously implanted into immunosuppressed (SCID or athymic) mice. Tumor (ca. 115 mm3) bearing animals (N = 10/group) were randomly allocated to receive the ADC treatments (at different doses) or the appropriate control (placebo included). Treatments were administered weekly for 5 consecutive weeks. Tumor growth (as median/group) was calculated 2-3 times per week. Twenty-four hours after the first dose, representative tumors were dissected free and processed for HER2 expression (erb2) and chromatin organization (Hoescht 33258). The treatment with MI130004 induced a long lasting antitumor effect with statistically significant increases (P&amp;lt;0.05) in median survival time compared to placebo. The highest dose of MI130004 induced complete tumor remissions in mice bearing BT-474, JIMT-1, Gastric-008, N87, SK-OV-3 and A2780cis xenografts lasting up to 120, 60, 165, 63, 237 and 231 days, respectively. Also 24-h post-dosing, MI130004 induced a dose-dependent disappearance of HER2-expressing cells as well as mitotic aberrations, this last being consistent with the mechanism of action of PM050489. These results demonstrate that PM050489, a marine derived compound, is a novel and remarkable payload for the design of new ADCs with potential therapeutic anti-cancer properties. Citation Format: Pablo Aviles, Maria Jose Guillen, Juan Manuel Dominguez, Carlos M. Galmarini, Carmen Cuevas. MI130004, a new ADC with a payload of marine origin shows outstanding activity against HER2-expressing tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A147.

Abstract 353: In vivo PET imaging of HER2 expression with GE226: An 18F-labelled affibody molecule

Abstract Background: The assessment of HER2 expression in biopsies from primary lesions of breast cancer patients is a standard procedure to select patients for HER2-targeted therapies. However, in metastatic disease in which HER2 status can change, determination of HER2 expression is not standard procedure, which complicates patient management. Herein we report evidence for GE226 ([18F]FBA-ZHER2:2891), a highly specific HER2 targeted imaging agent that can determine HER2 expression levels in preclinical tumour models. We propose that GE226 can be progressed to clinical development for non-invasive determination of the HER2 status in recurrent breast cancer patients to improve the clinical management and therapy selection. Methods: The highly selective HER2 targeted 18F-labelled Affibody molecule GE226 was characterized in a murine dual tumour breast cancer model bearing NCI-N87 (high HER2 status) and A431 (low HER2 status) xenografts in separate flanks. Tumour-bearing mice were injected with 3 to 10 MBq of GE226, followed by biodistribution or positron emission tomography (PET) imaging analysis. Results: Biodistribution analysis demonstrated good differentiation of GE226 retention between high and low HER2 expressing tumours (8.4% ID/g and 3.4% ID/g respectively, 30 min post injection). GE226 cleared quickly from background tissue, including kidneys, with excellent ratios for tumour-to-muscle (8.9 for high HER2 status tumours and 3.6 for low HER2 status tumours, 30 min post injection) and tumour-to-blood (2.5 for high HER2 status tumours and 1.0 for low HER2 status tumours, 30 min post injection). PET imaging of GE226 in the dual tumour mouse model showed a marked difference in signal intensity between the two tumour types. Conclusions: The highly selective HER2 targeted Affibody molecule GE226 can image different levels of HER2 expression in a dual-tumour preclinical model of breast cancer with good target-to-background ratios. These data compare favourably with previous patient SPECT and PET studies using the 111In- or 68Ga-labelled HER2-binding Affibody molecule ABY-002 (Baum et al., J Nucl Med. 2010;51(6):892-7) which supports the efficacy of this class of Affibody tracer for visualization of HER2 expressing metastases. We plan to progress GE226 further to assess HER2 status in metastatic breast cancer patients in clinical PET studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 353. doi:1538-7445.AM2012-353

Block Copolymer Micelles Target Auger Electron Radiotherapy to the Nucleus of HER2-Positive Breast Cancer Cells

Intracellular trafficking of Auger electron emitting radionuclides to perinuclear and nuclear regions of cells is critical to realizing their full therapeutic potential. In the present study, block copolymer micelles (BCMs) were labeled with the Auger electron emitter indium-111 ((111)In) and loaded with the radiosensitizer methotrexate. HER2 specific antibodies (trastuzumab fab) and nuclear localization signal (NLS; CGYGPKKKRKVGG) peptides were conjugated to the surface of the BCMs to direct uptake in HER2 expressing cells and subsequent localization in the cell nucleus. Cell uptake and intracellular distribution of the multifunctional BCMs were evaluated in a panel of breast cancer cell lines with different levels of HER2 expression. Indeed cell uptake was found to be HER2 density dependent, confirming receptor-mediated internalization of the BCMs. Importantly, conjugation of NLS peptides to the surface of BCMs was found to result in a significant increase in nuclear uptake of the radionuclide (111)In. Successful nuclear targeting was shown to improve the antipoliferative effect of the Auger electrons as measured by clonogenic assays. In addition, a significant radiation enhancement effect was observed by concurrent delivery of low-dose MTX and (111)In in all breast cancer cell lines evaluated.

Abstract 5284: 68Ga-radiolabeled DOTA-affibody molecule for in vivo assessment of HER2 expression with PET

Abstract Overexpression of HER2, a transmembrane tyrosine kinase receptor, is correlated with poor prognosis in patients with breast cancer. HER2 expression may vary between primary tumors and metastatic lesions and also change during the treatment. Therefore, there is a need for methods that can assess global HER2 expression non-invasively and reproducibly. PET imaging is well suited to this task based on its high sensitivity. Fluorine-18 (18F) is the most commonly used PET emitter for in vivo imaging since it is relatively short lived radionuclide (109 min). However, its production requires a cyclotron. Gallium-68 (68Ga) is an attractive alternative to 18F. It has a similarly short half-life (68 min) but can be obtained from a generator. In this work, we sought to monitor HER2 expression in vivo with 68Ga-labeled DOTA-ZHER2:2891 Affibody in a panel of breast cancer xenografts with different levels of HER2 expression. ZHER2:2891 Affibody molecules were conjugated with the chelator, DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid), and labeled with 68Ga. The in vitro binding characteristic of the tracer was evaluated using saturation cell-binding assay. In vivo studies were conducted in athymic nude mice bearing subcutaneous human breast cancer tumors with four different levels of HER2 expression. PET/CT scans were performed 1, 2, 3 hour post tracer injection. After imaging, the animals were sacrificed and selected organs were dissected to characterize the relationship between the signal detected by PET, ex vivo assessment of the tracer uptake, and the HER2 expression (in tumors). Nonspecific uptake was analyzed 1 hour post His6-ZTaq-GS-Cys-DOTA-68Ga (non-HER2-specific Affibody molecules) injection. In vitro assays showed high binding affinity of 68Ga-DOTA-ZHER2:2891 Affibody probe to MDA-MB-361 cells (Kd = 1.4±0.19 nM). In vivo biodistribution and PET imaging studies demonstrated high radioactivity uptake in the HER2 overexpressing tumors. Tracer was eliminated quickly from the blood and normal tissues, providing high tumor-to-blood ratios. The highest normal tissue concentration of radioactivity was seen in the kidneys. High-contrast PET images were recorded in BT474 and MDA-MB361 tumors 1 h post tracer injection. Low, but still detectable, uptake was observed for MCF7 tumors. Very good correlation was observed between PET and ex vivo estimates of tracer concentration and the receptor expression in the tumor tissue, as assessed by, respectively, radioactivity accumulation and enzyme-linked immunosorbent assay (ELISA). Stability tests of 68Ga-DOTA- ZHER2:2891 Affibody in vivo revealed that the tracer is stable in blood. These results suggest that PET imaging using 68Ga-DOTA-ZHER2:2891 Affibody is sensitive enough to detect different levels of HER2 expression in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5284. doi:10.1158/1538-7445.AM2011-5284

Abstract LB-323: Subclassification of squamous cell carcinomas of the head and neck based on HER/ErbB and c-MET receptor protein expression and activation profiles

Abstract The response rates of HER1 antibody monotherapy (∼13%) are comparable to the added response rates of HER1 antibody therapy when used in combination with chemotherapy (∼16%) in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck (SCCHN). Measures of HER1/ErbB1/EGFR protein levels by immunohistochemistry (IHC) or gene copy number by fluorescent in-situ hybridization (FISH) do not correlate with response, therefore, alternative gene or protein expression signatures are needed to identify patients most likely to respond to HER1 targeted therapies. Previously, quantitative VeraTag proximity immunoassays performed on formalin-fixed, paraffin imbedded SCCHN tumor tissue identified an activated HER1 signature (HER1-HER1 homodimers; HER1pY1173) that was significantly more pronounced in SCCHN compared to other carcinoma of the head and neck. In this study, we analyzed the expression of HER1, 2, 3 and c-MET along with the activation of HER1 and HER3 in 56 SCCHN tumors. Our results identified two distinct protein expression profiles: an activated HER1 profile, and a second profile characterized by the different expression levels of HER2, HER3, phosphorylated HER3, and c-MET relative to activated HER1. Our working hypothesis is that the first profile correlates with sensitivity to HER1 targeted therapies while the second profile correlates with either sensitivity or resistance to HER1 targeted therapies. HER1 protein levels measured by VeraTag assay varied over a ∼20-fold dynamic range, and significantly correlated with HER1 IHC staining and mRNA levels determined by qRT-PCR. HER2 and HER3 protein expression spanned a 15- to 20-fold range, and correlated with mRNA levels. Several of the highest HER1 expressing tumors have increased gene copy number; however, similar high values were measured in tumors without amplification, indicating that additional mechanisms of high HER1 expression exist in SCCHN. Although high HER1 activation measured by HER1-HER1 dimer and HER1pY1173 correlates with HER1 levels (r=∼0.6, p&amp;lt;0.05), and are associated with the highest levels of HER1 expression measured by multiple methods, not all high HER1 expressing tumors displayed highly activated HER1. These observations may explain why total HER1 expression in SCCHN tumors, determined by IHC or FISH, does not correlate with response to targeted therapies. On the other hand, approximately 16% of the SCCHN tumors studied here (9/56) exhibit a highly activated HER1 profile; similar in magnitude to the response rate seen with HER1 targeted antibody therapy. Future studies are designed to further validate the HER1 activation signature as a predictor of response to HER1 targeted therapies and/or the attenuation of response due to HER2, HER3 or c-MET expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-323. doi:10.1158/1538-7445.AM2011-LB-323